Developmental enhancement of AMP signaling and intracellular distribution of AMPKα2 and the activated form p-AMPKα(Thr172) during stem cell cardiac differentiation.

<p><b>A</b> - Compared to ES cells, derived CM have significantly higher total cellular ATP, ADP and free AMP levels. <b>B</b> - The ATP/ADP ratio was lower while the AMP/ATP ratio was markedly increased in CM compared to ES cells. <b>C</b> - mRNA levels of <i>Prkaa2</i>, the gene encoding the major cardiac AMPKα2 catalytic subunit, increased >3-fold upon cardiac differentiation while mRNA levels for <i>Prkaa1,</i> encoding the α1 catalytic subunit of AMPK, were down regulated. Protein level of AMPKα2, determined by Western blots (WB) and normalized to α-tubulin amount, was more abundant in CM. The amount of phosphorylated p-AMPKα(Thr172) was not significantly different between ES and CM. <b>D</b> - Immunocytochemical confocal microscopy images of AMPKα2 (green) distribution in ES-derived CM and intercalation with myofibrils (α-actinin, red). <b>E</b> – Co-immunostaining of CM for AMPKα2 and p-AMPKα(Thr172) indicated significant compartmentalization of AMP signaling with p-AMPKα(Thr172) localized predominantly in nuclei and concentrated in specific nuclear zones (inset). Representative images of n = 5–7. All scale bars are 10 µm.